Borrelia burgdorferi, the causative agent of Lyme disease, is maintained in nature through an infectious cycle that alternates between various species of small mammals and a tick vector. B. burgdorferi has an unusual segmented genome that includes a large number of linear and circular plasmids. Increasing evidence indicates that plasmid-encoded genes are critical for successful adaptation by the spirochete to the different environments that it encounters during its infectious cycle. We have developed genetic tools that we use to investigate basic aspects of the unusual genomic organization, cellular structure and metabolism of the Lyme disease spirochete. We have extended this investigation to an in vivo setting with an experimental system that closely mimics the natural arthropod vector/rodent host infectious cycle. Through an understanding of the basic molecular biology of the organism, we hope to gain insight into the infectious strategy utilized by this significant vector-borne pathogen and thereby facilitate efforts to prevent, diagnose and treat Lyme disease. The B. burgdorferi genome contains a linear chromosome and over 20 linear and circular plasmids, some of which carry essential genes. More than half of all plasmid-borne genes have no homologs outside of Borrelia and many of the predicted open reading frames on the chromosome encode novel proteins. Studies aimed at investigating gene function typically include inactivation of the gene of interest, assessment of phenotypic changes, and complementation of the mutation to determine if the wild type phenotype is restored. A standard method of complementation is reintroduction of a wild type copy of the inactivated gene on a shuttle vector. Shuttle vectors are available for complementation in B. burgdorferi, but they have a higher copy number than endogenous plasmids and can be lost in the absence of selection. In FY2015 we examined the use of the non-essential bbe02 locus on the in vivo essential linear plasmid 25 (lp25) for complementation in B. burgdorferi (1). We constructed a multi-purpose allelic exchange vector that contains bbe02 proximal sequences flanking a multiple cloning site and either of two selectable markers. There are significant advantages to this approach for complementation in B. burgdorferi. The bbe02 locus encodes a restriction-modification system that degrades foreign DNA, and inactivation of bbe02 has been shown to enhance genetic manipulation of B. burgdorferi. Thus, complementation by insertion into bbe02 facilitates subsequent transformations of B. burgdorferi, such as introduction of a reporter gene. A second advantage is the ensured retention of the in vivo essential lp25 during in vitro propagation of genetically manipulated spirochetes, through the concomitant insertion of a selectable marker during complementation at bbe02. Because lp25 carries genes essential for spirochete survival in both the tick vector and the mammalian host, complementation on lp25 ensures gene retention of the complementation construct throughout the in vivo experimental infectious cycle. Conveniently, complementation using this multi-purpose allelic exchange vector eliminates the design and construction of a new complementation plasmid for each mutation. Finally, complementation on lp25 will not alter the original mutation, as would be the case with performing in cis complementation. Environmental changes that accompany tick feeding are sensed by B. burgdorferi and utilized as cues to induce critical switches in gene expression and protein synthesis. This response includes the induction of outer surface protein C (OspC), a hallmark of adaptation to the mammalian environment. We previously established that OspC is required by B. burgdorferi to initiate infection of the mammalian host, but OspC is subsequently targeted by the host's acquired immune response and therefore ospC gene expression must be down-regulated for persistent infection. In FY2015 we determined that complementation of an ospC mutant using the multi-purpose allelic exchange construct described above restored OspC production and spirochete infectivity in a mouse model (1). Complementation in trans on a shuttle vector or elsewhere in the genome can alter the copy number and impact expression of the re-introduced gene relative to its native locus. Borrelia has both linear and circular replicons, and the form of the DNA on which a gene is located may also influence its expression. To address these potential concerns, we compared basal and induced expression of the ospC gene from its native locus on circular plasmid cp26 to that of the ospC gene introduced by allelic exchange on lp25. We found that mutant strains complemented with ospC on lp25 exhibited similar patterns of ospC transcription as the WT strain. Furthermore, we determined that the ospC gene carried by lp25 was stably retained by B. burgdorferi during persistent mouse infection. These FY2015 studies demonstrate the utility of targeting an endogenous plasmid of B. burgdorferi for stable in trans gene complementation and present an important new tool for molecular genetic investigations of the Lyme disease spirochete. (1) Irene N. Kasuma, Aaron Bestor, Kit Tilly and Patricia Rosa. 2015. Use of an endogenous plasmid for stable in trans complementation in Borrelia burgdorferi. Applied and Environmental Microbiology, Vol 81, no. 3 1038-1046.